Proteolysis resistant active vegf

ABSTRACT

The invention relates to endothelial growth factor (VEGF) in which the alanine at AA position 111 is replaced by proline. The arginine at AA position 110 may moreover be replaced by another amino acid. The invention also relates to derivatives of the VEGF according to the invention, nucleic acids, expression systems, medicaments and the use of the VEGF mutants of the invention for the treatment of chronic wounds.

The invention relates to vascular endothelial growth factor (VEGF) inwhich the alanine at AA position 111 is replaced by proline. Thearginine at AA position 110 may moreover be replaced by another aminoacid. The invention also relates to derivatives of the VEGF according tothe invention, nucleic acids, expression systems, medicaments and theuse of the VEGF mutants of the invention for the treatment of chronicwounds.

An important stage in cutaneous wound healing is the formation of agranulation tissue. Firstly associated with the latter is the migrationin of newly formed vessels (neoangiogenesis). Numerous experimental andclinical studies show that chronic wounds are characterized by impairedangiogenesis and thus diminished formation of granulation tissue.

A large number of mediators which stimulate angiogenesis during woundhealing are known. They include firstly the factors which, besidesstimulating endothelial cells, also activate mesenchymal and/orepidermal cells (bFGF, aFGF, TGF-a, PDGF), and secondly so-calledendothelial cell-specific factors whose receptors are substantiallyconfined to endothelial cells (VEGF, angiopoietin). A large number ofphysiological and pathological reactions involving the blood vesselscorrelates with an increased expression of VEGF and its receptors, sothat VEGF assumes a central role in angiogenesis of the skin. The firstindications of the possible importance of VEGF in wound healingimpairments were provided on the basis of experiments on VEGF expressionin diabetic mice (db/db mice) (Frank et al. 1995). It was possible toshow in this model that the wound healing impairment correlates with adiminished VEGF expression. It has recently been possible to providesupport for the role of VEGF in wound healing by a further transgenicanimal model (Fukumura et al., 1998) and detection of VEGF in the wounddischarge from acute human wounds (Nissen et al., 1998).

It has further been shown that there is increased expression of the mRNAof VEGF and its receptors in the tissue of chronic wounds (Lauer et al.,2000). Investigations by SDS-PAGE show, however, breakdown of the VEGFprotein in the chronic wound environment, in contrast to the acutewound. This breakdown leads to a significant loss of the biologicalactivity and may thus, despite the increased expression of the VEGFreceptors, underlie a deficient stimulation of neoangiogenesis in thechronic wound environment. As explained above, it was possible to showthat plasmin is involved in the cleavage of VEGF in the chronic woundenvironment (Lauer et al., 2000).

Cleavage of VEGF₁₆₅ via plasmin leads to detachment of thecarboxyl-terminal domain which is encoded by Exon 7. Whereas Exons 3 and4 determine the binding properties of VEGF to the VEGF receptors Flt-1and Flk-1/KDR, Exon 7 has a critical importance in the interaction ofVEGF with neuropilin-1 (Keyt et al. 1996). Neuropilin-1 is a 130 kDacell surface glycoprotein. Its role in the potentiation of the mitogeniceffect of VEGF on endothelial cells was described only recently (Sokeret al. 1998). In this connection, the interaction of neuropilin-1 withFlk-1/KDR appears to be important because binding solely of VEGF toneuropilin-1 has no signal effect.

Plasmin belongs to the class of serine proteases. These enzymes are ableto cleave peptide linkages. The cleavage takes place by a so-calledcatalytic triad. In the catalytic centre thereof an essential part isplayed in particular by the eponymous serine, but also by the aminoacids histidine and aspartate, because the process of peptide cleavagetakes place by means of them (Stryer 1987, pp. 231 et seq.). Althoughthe mechanism of the linkage cleavage is identical in all serineproteases, they differ markedly in their substrate specificity. Thus,plasmin, just like trypsin, cleaves peptide linkages after the basicamino acids lysine and arginine. However, the substrate specificity ofplasmin, which is determined by the structure of the catalytic centre,leads to plasmin being unable to cleave all these linkages. Catalysis ofpeptide-linkage cleavage is possible only if the corresponding proteinsegments are able to interact with the catalytic centre of the enzyme(Powers et al. 1993; Stryer 1987). To date, no unambiguous consensussequence of a plasmin cleavage site is known.

The present invention is based on the object of providing improved meansfor healing chronic wounds. Surprisingly, this object is achieved by theprovision according to the invention of a vascular endothelial growthfactor (VEGF) variant which is characterized in that at least one aminoacid in the sequence of the native vascular endothelial growth factor atpositions 109 to 112 of the native vascular endothelial growth factor isreplaced by another amino acid or a deletion.

In one embodiment of the invention, at least one amino acid in thesequence of the native vascular endothelial growth factor is replaced byproline in the VEGF variant according to the invention the positions 109to 112. In a further embodiment, besides proline, at least one furtheramino acid at one of positions 109 to 112 in the VEGF according to theinvention is replaced or a deletion.

In a further embodiment, the alanine at AA position 111 of the nativevascular endothelial growth factor is replaced by proline in the VEGFvariant according to the invention.

In another embodiment, the arginine at AA position 110 of the nativevascular endothelial growth factor is replaced by another amino acid inthe VEGF variant according to the invention. In particular, the arginineat AA position 110 of the native vascular endothelial growth factor isreplaced by proline.

In a further embodiment of the invention, the arginine at AA position111 of the native vascular endothelial growth factor is replaced byanother amino acid in the VEGF.

It is possible in particular for the arginine at AA position 111 of thenative vascular endothelial growth factor and the alanine at AA position111 of the native vascular endothelial growth factor to be replaced byproline in the VEGF variant according to the invention.

The VEGF mutants according to the invention are preferably in the formof one of the splice variants VEGF₁₂₁, VEGF₁₄₅, VEGF₁₆₅, VEGF₁₈₃,VEGF₁₈₉ or VEGF₂₀₆.

The VEGF mutants according to the invention display not only markedlyincreased stability towards plasmin, but also an activity comparable tothat of wild-type VEGF. Surprisingly, the VEGF variants according to theinvention additionally display distinctly increased stability in chronicwound fluids.

The mutations have been carried out at a site which is critical for thebiological activity of the VEGF molecule. There was thus a fear that achange in the protein structure in this region has a negative effect onthe activity of VEGF₁₆₅. The amino acid proline, which is introducedaccording to the invention at position 111, is a cyclic α-imino acid.Owing to the cyclic form of the pyrrolidine residue, it has a rigidconformation which also has an effect on the structure of the respectiveproteins. Thus, proline acts for example as a strong α-helix breaker. Itis therefore particularly surprising that replacement precisely ofalanine at position 111 by proline generates a VEGF mutant which isstable towards the protease plasmin, is stable in chronic would fluidsand, at the same time, still has an activity corresponding to that ofthe wild-type protein.

The invention relates in particular to VEGF variants of the twosequences Seq. No. 1 or Seq. No. 2.

The invention also relates to variants of the VEGF mutants mentionedabove, in which the amino acid sequences are modified or derivatized, orcomprise mutations, insertions or deletions. This relates in particularto VEGF variants in which further single amino acids are replaced, andthose which are glycosylated, amidated, acetylated, sulphated orphosphorylated. Such VEGF variants preferably have an activitycomparable to or higher than the wild-type VEGF.

The VEGF variants according to the invention may also have a signalsequence. The signal sequence may be connected N-terminally to the aminoacid chain of the VEGF variant and have the sequence Met Asn Phe Leu SerTrp Ser Val His Trp Ser Leu Ala Leu Leu Leu Tyr Leu His His Ala Lys TrpSer Gln Ala.

The invention also relates to nucleic acids which code for theabovementioned VGEF mutants, and vectors for VEGF expression whichcomprise such nucleic acids.

The invention relates to a medicament which comprises the abovementionedmutants of VEGF, and to the use of the VEGF mutants for producing amedicament for the treatment of chronic wounds, caused by vascularlesions such as chronic venous insufficiency (CVI), primary/secondarylymphoedema, arterial occlusive disease, metabolic disorders such asdiabetes mellitus, gout or decubitus ulcer, chronic inflammatorydisorders such as pyoderma gangrenosum, vasculitis, perforatingdermatoses such as diabetic necrobiosis lipoidica and granulomaannulare, haematological primary disorders such as coagulation defects,sickle cell anaemia and polycythemia vera, tumours, such as primarycutaneous tumours and ulcerative metastases, and for plasmin inhibition,for inducing neoangiogenesis and/or for inhibiting matrix degradation.

Topical use of growth factors represents a novel therapeutic concept inwound healing. It has been possible to observe an improvement in thehealing of chronic wounds in a large number of clinical studies with theuse of EGF, bFGF, PDWHF and PDGF (Scharffetter-Kochanek et al. 2000).However, a criticism which should be noted is that the results of thesestudies did not come up to the expectations which existed in view of thegood activity of these mediators in animal models (Lawrence et al.1994). This restricted activity of the growth factors is certainlysubstantially explained by the increased proteolytic activity in thechronic wound environment, which leads to degradation of the topicallyapplied factors. It is thus clear that local wound management byadministration of growth factors represents a promising noveltherapeutic strategy. However, it is necessary to develop strategieswhich control the proteolytic activity in the chronic wound environment.The production of master cytokines with increased stability in thechronic wound environment certainly represents a novel therapeuticapproach in this connection. The VEGF mutants according to the inventionare particularly suitable, because of their high stability in the woundfluid, for the topical treatment of chronic wounds.

EXEMPLARY EMBODIMENT

Mutagenesis:

Four mutants were produced by site-directed mutagenesis by carrying outtargeted amino acid replacements at Arg₁₁₀ and Ala₁₁₁. The cDNA whichcodes for human VEGF₁₆₅ was cloned into the SV40 replication expressionvector pcDNA 3.1 (from Invitrogen, De Schelp, NL) using the BamHI andEcoRI cleavage sites in the cloning site. The Gene Editor™ system fromPromega (Mannheim) was used for the site-directed in vitro mutagenesis.This system is based on annealing of oligonucleotides which harbour theappropriate mutation onto the initial sequence. The initial sequence ofVEGF₁₆₅ in the region of the mutations is:    106 107 108 109 110 111112 113 GA CCA AAG AAA GAT AGA GCA AGA CAA G    Pro Lys Lys Asp Arg AlaArg Gln

To introduce the mutations, the following mismatch oligonucleotides wereused as primers: Mutation 1: Mut_(Ala): GA CCA AAG AAA GAT GCC GCA AGACAA G    Pro Lys Lys Asp Ala Ala Arg Gln Mutation 2: Mut_(Gln): GA CCAAAG AAA GAT CAG GCA AGA CAA G    Pro Lys Lys Asp Gln Ala Arg GlnMutation 3: Mut_(Pro): GA CCA AAG AAA GAT AGG CCA AGA CAA G    Pro LysLys Asp Arg Pro Arg Gln Mutation 4: Mut_(Lys-Pro): GA CCA AAG AAA GATAAG CCA AGA CAA G    Pro Lys Lys Asp Lys Pro Arg Gln

The mutagenesis primers used are each detailed with the modified aminoacid sequences obtained therewith. The regions with the bases or aminoacids which are changed from the wild-type sequence are in italics.

In mutation 1, arginine₁₁₀ was replaced by a nonpolar alanine. Inmutation 2, a polar, uncharged glutamine was introduced at the sameposition. In mutant 3, the alanine at position 111, not the basicarginine₁₁₀, was replaced by a proline. In mutant 4, two amino acidswere replaced. In this case, lysine and proline were introduced in placeof arginine₁₁₀ and alanine₁₁₁. After the mutagenesis had been carriedout, the mutations were verified by sequence analysis. The resultingVEGF mutants had the following sequences for amino acids 109-112:VEGF₁₆₅ wild type: -Asp₁₀₉Arg₁₁₀Ala₁₁₁Arg₁₁₂- Mut_(Gln):-Asp₁₀₉Gln₁₁₀Ala₁₁₁Arg₁₁₂- Mut_(Ala): -Asp₁₀₉Ala₁₁₀Ala₁₁₁Arg₁₁₂-Mut_(Pro): -Asp₁₀₉Arg₁₁₀Pro₁₁₁Arg₁₁₂- Mut_(Lys-Pro):-Asp₁₀₉Lys₁₁₀Pro₁₁₁Arg₁₁₂-

The mutants Mut_(Pro) and Mut_(Lys-Pro) are mutants according to theinvention, whereas Mut_(Gln) and Mut_(Ala) are produced and investigatedfor the purposes of comparison. The resulting VEGF₁₆₅ expression vectorswere used in the further investigations.

Production of Recombinant VEGF₆₅ Protein

VEGF₁₆₅ protein was expressed in eukaryotic COS-1 cells. The pcDNA 3.1expression vector used comprises an SV-40 origin of replication. Thisserves to amplify the vector in cells which express a large T antigen ofthe SV-40 virus. The COS-1 cells used possess a corresponding elementintegrated into the genome, so that episomal replication of the vectorresults. Expression of the target protein VEGF for several days isachieved thereby without stable integration (transformation) of thevector into the cell genome. The COS-1 cells were transfected with theexpression plasmids obtained in the mutagenesis. For this purpose, theSuperfect transfection reagent (QIAGEN, Hilden) was used according tothe manufacturer's protocols.

Like a large number of growth factors, VEGF₁₆₅ also has aheparin-binding site which is located at the basic C terminus. Thebinding to heparin was exploited for purification of the protein byaffinity chromatography (Mohanraj et al. 1995). The VEGF and VEGFvariants were isolated by the following steps:

The COS-1 cells transformed with the expression plasmids were cultivatedin serum-free DMEM (Dulbecco's modified Eagle's medium) comprising 10%fetal calf serum (FSC), 2 mM L-glutamine, penicillin (10 U/ml) andstreptomycin (10 μg/ml) and ITS supplement (Sigma, Deisenhofen).Conditioned medium (200 ml) was collected after 48 h and incubated with5 ml of heparin-Sepharose (Pharmacia, Freiburg) at 4° C. for 4 hours.The heparin-Sepharose was packed into a column. The latter was loadedwith culture medium at a flow rate of 25 ml/h. The following steps werecarried out:

A: Affinity chromatography with heparin-Sepharose

1. Washing: 0.1 M NaCl; 20 mM Tris/pH 7.2

2. Washing: 0.3 M NaCl; 20 mM Tris/pH 7.2

3. Elution: 0.9 M NaCl; 20 mM Tris/pH 7.2

B: Analysis of the resulting fractions by Western blot analysis

C: Desalting of the VEGF-containing fractions by gel filtration

Running buffer: 10 mM Tris/pH 7.2

D: Lyophilization of the solution and determination of the concentrationby ELISA

The resulting VEGF was investigated by SDS-PAGE. The VEGF proteinobtained from COS-1 cells differs in its migration behaviour in SDS-PAGEfrom the commercially available VEGF₁₆₅ protein used (from R&D Systems).In addition to the signal to be detected at 42 kDA (FIG. 1, lane 6), afurther band with a molecular weight which is a few kDA higher is alsoevident. The reason for this double band of the VGEF protein expressedin COS-1 cells is an altered glycosylation of the growth factor. Onexpression of VEGF in COS-1 cells there is formation of two differentlyglycosylated proteins. One form (42 kDa) is identical in itsglycosylation to the recombinant VEGF₁₆₅ which has been used to date andwhich was produced in insect cells using a baculovirus expression system(R&D Systems, FIG. 1, lane 1). It has an N-glycosylation on the aminoacid asparagine at position 74 (Gospodarowicz et al. 1989; Keck et al.1989). The second band at a higher molecular weight (45 kDa) resultsfrom further glycosylation of the protein. The difference in theglycosylation is known for expression in COS cells and has no effect onthe biological activity of the growth factor (R&D Systems).

Characterization of the Biochemical and Biological Properties of thePurified VEGF₁₆ Proteins

I. Analysis of the Stability of the VEGF₁₆₅ Proteins and its Mutations:

a) Incubation in Plasmin

The four purified mutated VEGF proteins were initially investigated fortheir stability towards the protease plasmin. It was investigatedwhether the mutations carried out lead to an altered degradationbehaviour compared with wild-type VEGF.

FIG. 1 shows the results of incubation of the VEGF wild type and theVEGF mutants with plasmin. Incubation of the VEGF wild type synthesizedin COS-1 cells (A, lane 6-10) shows degradation of the growth factorafter only 15 minutes. In this case, accurate determination of the sizeof the resulting fragments by SDS-PAGE is difficult because the signalsoverlap with the two bands of the differently glycosylated protein.However, the degradation pattern is similar to that of the commerciallyobtainable VEGF₁₆₅ (FIG. 1A, lane 1-5). Thus, a fragment with amolecular weight of 38 kDA can be detected after 45 minutes. Thiscorresponds to the 110 dimer fragment of the less glycosylated VEGFvariant. These results clearly show that the VEGF protein expressed inthe COS-1 cells is also cleaved by plasmin under the chosen conditions.

FIG. 1B (lane 1-17) shows the results of incubation of mutated proteins.Incubation of the arginine to alanine mutation is shown first (lane1-5). At zero incubation time, two bands are detectable for thedifferently glycosylated variants of the VEGF protein, as with the wildtype. However, in this case, because of the higher signal intensity,they cannot be differentiated from one another so clearly as with theVEGF₁₆₅ wild type. In contrast to the VEGF wild type, the mutatedprotein shows no change in the migration behaviour up to 240 minutesafter incubation.

This observation suggests that the arginine₁₁₀ to alanine₁₁₀ mutationhas led to inactivation of the plasmin cleavage site. As shown furtherin FIG. 1B, the three other mutants Mut_(Pro), Mut_(Gln) andMut_(Lys-Pro) also show a comparable stability of the signal bands at 45and 42 kDA after incubation with plasmid for 240 minutes. A control inwhich the VEGF₁₆₅ wild type was incubated with plasmin buffer at 37° C.for 4 hours is not degraded (lanes 18 and 19). Overall, theseexperiments indicate that the produced and purified VEGF mutants arestable towards the protease plasmin.

b) Incubation in Acute and Chronic Wound Fluid

In the next step, the degradation of the VEGF mutants in wound fluidfrom patients with acute and chronic wounds was analysed. On incubationof the VEGF₁₆₅ wild type and all VEGF mutants in acute wound fluid, nodegradation was detectable after 240 minutes.

FIG. 2 shows the effect of chronic wound fluid on the stability of theVEGF proteins. Incubation of the VEGF wild type synthesized in COS-1cells (FIG. 2A, lane 1-4) for 240 minutes shows degradation of thegrowth factor with a fragment of about 38 kDa. This corresponds to the110 dimer fragment of the less glycosylated VEGF variant.

In contrast to the wild type, the VEGF)₆₅ mutants show a differentdegradation behaviour on incubation in chronic wound fluid. On the onehand, the degradation process observed in the mutations Mut_(Gln) (FIG.2B, lanes 13-16 and Mut_(Ala) (lanes 5-8) is comparable to that of thewild type. Fragments with a molecular weight of about 38 kDa areproduced after only about 20 min.

On the other hand, analysis of the mutants Mut_(Pro) (lanes 9-12) andMut_(Lys-Pro) (Lanes 1-4, 17-20) shows a breakdown behaviour differentfrom the wild type and the mutants Mut_(Ala) and Mut_(Gln). A stablesignal at 42 and 45 kDa is seen in the SDS-PAGE up to 60 minutes afterincubation. This indicates stabilization of the mutated proteinsMut_(Pro) and Mut_(Lys-Pro) in the chronic wound fluid. This differencein the degradation behaviour of the mutants with neutral/nonpolar aminoacid and those with proline suggests that further proteases, besidesplasmin, are involved in the breakdown of VEGF in the chronic woundenvironment.

Degradation is observable with all mutated proteins 240 minutes afterincubation in chronic wound fluid. In these cases there is not justformation of clearly defined breakdown fragments; on the contrary, adiffuse signal between 38 and 45 kDa appears after 240 min. Thispresumably involves proteolysis in the region of the first 20 aminoacids (recognition site of the antibody), because the signal strengthdecreases markedly after 240 min.

In summary, the results indicate that the VEGF mutants with proline atposition 111 are initially stabilized in chronic wound fluid but aredegraded in the long term. Comparable results were observed in the wouldfluids from three different patients with chronic venous insufficiency.The experiments for the various wound fluids were repeated at leasttwice (FIG. 2B: patient X lanes 1-4; patient Y lanes 17-20). Theresulting band pattern always remained the same moreover.

FIG. 2C shows a densitometric evaluation of the breakdown of VEGF wildtype and Mut_(Lys-Pro). The aim of the investigation was to quantify thestabilization of the VEGF mutant in the chronic wound fluid. For thispurpose, the time-dependent change in the signal strength at the levelof the initial signal (42-45 kDa region) compared with the signal attime zero was determined. The densitometric densities measured at thevarious times are depicted as percentage of the initial signal. It isclear in this densitometric investigation that at every measurement timethe VEGF mutant shows a stronger signal by comparison with the VEGF wildtype in the 42-45 kDa region, and thus intact VEGF₁₆₅ protein ispresent. This observation suggests that this mutation leads to animproved stability and bioactivity of the VEGF protein in the chronicwound environment. The difference between wild type and mutant isstatistically significant only 20 minutes after the incubation. Themeasurements were carried out with identical wound fluid for threeindependent experiments.

II. Investigations of the Biological Activity of VEGF₁₆₅ Wild Type andthe Mutated Variants:

It was investigated whether the mutations have an effect on thebiological activity of the VEGF molecule. The biological activity wasassayed by means of a BrdU proliferation assay (Roche Diagnostics,Mannheim) on human umbilical vein endothelial cells (HUVE cells) inaccordance with the manufacturer's information. This entailed the HUVEcells being cultivated with addition of various VEGF mutants, thenincubated with BrdU solution for 6 hours and fixed, after which an ELISAwas carried out using a BrdU-specific antibody.

VEGF concentrations between 1 ng/ml and 25 ng/ml were employed.Commercially available recombinant VEGF₁₆₅ protein (R&D Systems) andVEGF₁₆₅ wild type synthesized in COS-1 cells showed a half-maximumstimulation of BrdU incorporation at about 3 ng/ml (FIG. 3). The mutatedVEGF proteins are characterized by a stimulation of endothelial cellproliferation which is comparable to the VEGF wild type synthesized inCOS-1 cells. The maximum stimulation of all the proteins synthesized inCOS-1 cells was less than that by commercially obtainable VEGF₁₆₅ wildtype. The reason for the difference between the two curved profiles maybe the different expression systems and purification methods for theproteins (Mohanraj et al. 1995). The biological activity of VEGF₁₆₅ isthus not significantly affected by the mutations carried out.

The question of the extent to which the biological activity of theVEGF₁₆₅ wild type and of the VEGF mutants is affected after plasminincubation was subsequently examined. For this purpose, the VEGFproteins were incubated with plasmin, and then the biological activitywas investigated by means of a BrdU proliferation assay on HUVE cells.

In the graphical representation (FIG. 4), the BrdU incorporation isshown as percentage of the initial signal (time t=0). Incubation of theVEGF wild types (synthesized in COS-1 cells and from R&D Systems) and ofthe VEGF mutants Mut_(Ala) and Mut_(Lys-Pro) in plasmin buffer at 37° C.shows no impairment of the biological activity of the proteins (FIG.4A-D). In contrast thereto, incubation of the VEGF₁₆₅ wild types inplasmin leads to a marked reduction in the biological activity (FIG. 4A,B). An activity loss of at least 20% is seen only 20 minutes afterincubation, and then falls further to about 50% of the initial activityafter 240 minutes. The Mut_(Ala) and Mut_(Lys-Pro) mutants show nosignificant activity loss after incubation with plasmin (FIG. 4C, D).These results underline the “plasmin resistance” of the mutantsdemonstrated in the Western blot (FIG. 1) and show that the mutatedproteins are stable even after incubation with plasmin.

The introduced mutations thus result in an inhibition of VEGF cleavageby plasmin. A stabilization of VEGF and thus an increased biologicalactivity in the chronic wound environment can be brought about by theAla₁₁₁ to Pro₁₁₁ mutation.

FIG. 1: The VEGF₁₆₅ mutations are resistant to cleavage by plasmin. Thefigure shows incubation of VEGF₁₆₅ and the mutated proteins in a plasminsolution [0.01 U/ml] or buffer solution (B, lanes 18, 19) for the statedperiods. Analysis of the degradation behaviour took place by Westernblotting and immunodetection.

FIG. 2: The Ala₁₁₁ to Pro₁₁₁ mutation increases the stability of VEGF inchronic wound fluid. A) VEGF₁₆₅ wild type expressed in COS-1 cells andB) the VEGF variants were incubated in chronic wound fluid for thestated periods, and the degradation behaviour was visualized byimmunodetection. In this case, wound fluids from two different patientswere investigated: patient X, lanes 1-16; patient Y: lanes 17-20). C)Densitometric visualization of the degradation of VEGF wild type andMut_(Lys-Pro) in chronic wound fluid. The relative signal strength fromthree independently performed Western blot analyses (mean+/−SD) isshown.

FIG. 3: The VEGF mutants are biologically active. VEGF₁₆₅ wild type andVEGF mutants were each incubated in increasing concentrations with HUVEcells. The rate of incorporation of the base analogue into the DNA ofthe proliferating cells determined by BrdU ELISA is shown (mean+/−SD;n=3).

FIG. 4: Plasmin does not alter the biological activity of the VEGF₁₆₅mutants. A comparison is shown of the relative BrdU incorporation intoHUVE cells through stimulation with VEGF₁₆₅ wild type (A, B), Mut_(Ala)(C) and Mut_(Lys) Pro (D) after incubation of the stated protein inbuffer or plasmin (means+/−SD; n=3).

REFERENCES

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1. A vascular endothelial growth factor (VEGF) variant, comprising anamino acid sequence, wherein said sequence is the amino acid sequence ofthe native vascular endothelial growth factor in which at least oneamino acid at positions 109 to 112 of the native vascular endothelialgrowth factor is replaced by another amino acid or is deleted.
 2. TheVEGF variant according to claim 1, wherein said at least one amino acidin the sequence of the native vascular endothelial growth factor atpositions 109 to 112 is replaced by proline.
 3. The VEGF variantaccording to claim 2, wherein at least one further amino acid at one ofthe positions 109 to 112 is replaced or is deleted.
 4. The VEGF variantaccording to claim 1, wherein an alanine at position 111 of the nativevascular endothelial growth factor is replaced by proline.
 5. The VEGFvariant according to claim 4, wherein an arginine at position 110 of thenative vascular endothelial growth factor is replaced by another aminoacid.
 6. The VEGF variant according to claim 1, wherein an arginine atposition 110 of the native vascular endothelial growth factor isreplaced by proline.
 7. The VEGF variant according to claim 6, whereinan alanine at position 111 of the native vascular endothelial growthfactor is replaced by another amino acid.
 8. The VEGF variant accordingto claim 5, wherein said other amino acid is a proline.
 9. The VEGFvariant according to claim 1, wherein the VEGF variant is in the form ofany of the splice variants VEGF₁₂₁, VEGF₁₄₅, VEGF₁₆₅, VEGF₁₈₃, VEGF₁₈₉or VEGF₂₀₆.
 10. The VEGF₁₆₅ variant according to claim 9, wherein theVEGF₁₆₅ variant has one of the amino acid sequences Seq. ID No. 1: AlaPro Met Ala Glu Gly Gly Gly Gln Asn His His Glu Val Val Lys Phe Met AspVal Tyr Gln Arg Ser Tyr Cys His Pro Ile Glu Thr Leu Val Asp Ile Phe GlnGlu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys Pro Ser Cys Val Pro Leu MetArg Cys Gly Gly Cys Cys Asn Asp Glu Gly Leu Glu Cys Val Pro Thr Glu GluSer Asn Ile Thr Met Gln Ile Met Arg Ile Lys Pro His Gln Gly Gln His IleGly Glu Met Ser Phe Leu Gln His Asn Lys Cys Glu Cys Arg Pro Lys Lys AspArg Pro Arg Gln Glu Asn Pro Cys Gly Pro Cys Ser Glu Arg Arg Lys His LeuPhe Val Gln Asp Pro Gln Thr Cys Lys Cys Ser Cys Lys Asn Thr Asp Ser ArgCys Lys Ala Arg Gln Leu Glu Leu Asn Glu Arg Thr Cys Arg Cys Asp Lys ProArg Arg or Seq. ID No. 2: Ala Pro Met Ala Glu Gly Gly Gly Gln Asn HisHis Glu Val Val Lys Phe Met Asp Val Tyr Gln Arg Ser Tyr Cys His Pro IleGlu Thr Leu Val Asp Ile Phe Gln Glu Tyr Pro Asp Glu Ile Glu Tyr Ile PheLys Pro Ser Cys Val Pro Leu Met Arg Cys Gly Gly Cys Cys Asn Asp Glu GlyLeu Glu Cys Val Pro Thr Glu Glu Ser Asn Ile Thr Met Gln Ile Met Arg IleLys Pro His Gln Gly Gln His Ile Gly Glu Met Ser Phe Leu Gln His Asn LysCys Glu Cys Arg Pro Lys Lys Asp Lys Pro Arg Gln Glu Asn Pro Cys Gly ProCys Ser Glu Arg Arg Lys His Leu Phe Val Gln Asp Pro Gln Thr Cys Lys CysSer Cys Lys Asn Thr Asp Ser Arg Cys Lys Ala Arg Gln Leu Glu Leu Asn GluArg Thr Cys Arg Cys Asp Lys Pro Arg Arg.


11. The VEGF variant according to claim 1, wherein the amino acid chainis modified or derivatized and/or comprises mutations, insertions and/ordeletions and/or has a signal sequence.
 12. The VEGF variant accordingto claim 11, wherein the signal sequence is connected N-terminally tothe amino acid chain of the VEGF variant and has the sequence of Seq. IDNo. 3: Met Asn Phe Leu Ser Trp Ser Val His Trp Ser Leu Ala Leu Leu LeuTyr Leu His His Ala Lys Trp Ser Gln Ala.


13. A nucleic acid encoding the VEGF variant of claim
 1. 14. Vectorscomprising nucleic acids according to claim 13 for expression of saidVEGF variant.
 15. A medicament comprising VEGF variants according toclaim
 1. 16. (canceled)
 17. A method for treating chronic woundscomprising administering to a patient in need thereof an effectiveamount of the VEGF variant of claim
 1. 18. A method for treating acondition induced by a vascular lesion comprising administering to apatient in need thereof an effective amount of the VEGF variant of claim1, wherein said condition is chronic venous insufficiency (CVI),primary/secondary lymphoedema, arterial occlusive disease, a metabolicdisorder, a chronic inflammatory disorder, a perforating dermatosis, ahaematological primary disorder, sickle-cell anaemia and polycythemiavera, or a tumor.
 19. The method of claim 18, wherein said metabolicdisorder is diabetes mellitus, gout or decubitus ulcer, said chronicinflammatory disorder is pyoderma gangrenosum or vasculitis, saidperforating dermatosis is diabetic necrobiosis lipoidica or granulomaannulare, said haematological primary disorder is a coagulation defectand said tumor is a primary cutaneous tumor or an ulcerative metastasis.20. A method for inhibiting plasmin, inducting neoangiogenesis, and/orinhibiting matrix degradation comprising administering to a patient inneed thereof an effective amount of the VEGF variant of claim 1.